Combinations for the treatment of inflammatory disorders

ABSTRACT

The invention features methods and compositions for the treatment of immunonflammatory disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Utility application Ser. No.10/191,149, filed Jul. 9, 2002, now U.S. Pat. No. 6,897,206 which claimsthe benefit of U.S. Provisional Application Ser. No. 60/304,089, filedJul. 9, 2001, each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The invention relates to the treatment of inflammatory disorders.

Inflammation occurs when tissues are injured by viruses, bacteria,trauma, chemicals, heat, cold or any other harmful stimulus. Chemicalsincluding bradykinin, histamine, serotonin and others are released,attracting tissue macrophages and white blood cells to localize in anarea to engulf and destroy foreign substances. During this process,chemical mediators such as TNFα are released, giving rise toinflammation. Inflammatory disorders are those in which the inflammationis sustained or chronic. One example of an inflammatory disorder isosteoarthritis.

Immunoinflammatory disorders (e.g., rheumatoid arthritis, psoriasis,ulcerative colitis, Crohn's disease, stroke-induced brain cell death,ankylosing spondylitis, fibromyalgia, and autoimmune diseases such asasthma, multiple sclerosis, type I diabetes, systemic lupuserythematosus, scleroderma, systemic sclerosis, and Sjögren's syndrome)are inflammatory disorders characterized by dysregulation of the immunesystem and inappropriate mobilization of body's defenses against its ownhealthy tissue.

One percent of humans world-wide are afflicted with rheumatoidarthritis, a relentless, progressive disease causing severe swelling,pain, and eventual deformity and destruction of joints. According to theArthritis Foundation, rheumatoid arthritis currently affects over twomillion Americans, of which women are three times more likely to beafflicted. Rheumatoid arthritis is characterized by inflammation of thelining of the joints and/or other internal organs, and the presence ofelevated numbers of lymphocytes and high levels of proinflammatorycytokines.

Treatment of rheumatoid arthritis generally includes administration of(i) non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen,diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam,nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin,celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodiumand magnesium salicylate); (ii) steroids (e.g., cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone); (iii) DMARDs, i.e., disease modifyingantirheumatic drugs (e.g., cyclosporine, azathioprine, methotrexate,leflunomide, cyclophosphamide, hydroxychloroquine, sulfasalazine,D-penicillamine, minocycline, and gold); or (iv) recombinant proteins(e.g., ENBREL® (etanercept, a soluble TNF receptor) and REMICADE®(infliximab) a chimeric monoclonal anti-TNF antibody).

There is a need to develop new regimens for the treatment of rheumatoidarthritis, and other inflammatory disorders.

SUMMARY OF THE INVENTION

We have discovered that the combination of amoxapine(2-cloro-11(1-piperazinyl)dibenz[b,f][1,4]oxapine) and prednisolone(also known as 1-dehydrocortisol; 1-dehydrohydrocortisone;1,4-pregnadiene-11beta, 17alpha, 21-triol-3,20-dione; and 11beta,17alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione) brings aboutsubstantial suppression of TNFα levels induced in peripheral bloodmononuclear cells (PBMCs).

Amoxapine is a tricyclic antidepressant (TCA). Based on the ability ofamoxapine to act in concert with prednisolone to inhibit TNFα levels,one skilled in the art will recognize that other TCAs can also be usedin the present invention. Structural analogs of amoxapine that are nottricyclic antidepressants can also be used. Exemplary structural analogsinclude, for example, clothiapine, perlapine, fluperlapine, and dibenz(b,f)(1,4)oxazepine, 2-chloro-11-(4-methyl-1-piperazinyl)-,monohydrochloride.

Prednisolone is a corticosteroid. Based on the shared structuralfeatures and apparent mechanism of action among the corticosteroidfamily, one skilled in the art will recognize that other corticosteroidscan be used in combination with amoxapine or other TCAs to treatinflammatory disorders.

Accordingly, the invention features a method for treating a patienthaving an inflammatory disorder, by administering to the patient (i) aTCA (e.g, amoxapine); and (ii) a corticosteroid (e.g., prednisolone)simultaneously or within 14 days of each other, in amounts sufficient tosuppress TNFα levels sufficiently to produce a therapeutic benefit tothe patient. In one embodiment, the two compounds are amoxapine andprednisolone.

The invention also features a method for treating a patient having aninflammatory disorder by administering to the patient (i) clothiapine,perlapine, fluperlapine, or dibenz (b,f)(1,4)oxazepine;2-chloro-11-(4-methyl-1-piperazinyl)-, monohydrochloride; and (ii) acorticosteroid (e.g., prednisolone) simultaneously or within 14 days ofeach other, in amounts sufficient to suppress TNFα levels sufficientlyto produce a therapeutic benefit to the patient.

Preferably, the two compounds of the invention are administered withinten days of each other, more preferably within five days of each other,and most preferably within twenty-four hours of each other, orsimultaneously. The disorder treated according to the invention can be,for example, rheumatoid arthritis, psoriasis, ulcerative colitis,Crohn's disease, stroke-induced brain cell death, ankylosingspondylitis, and fibromyalgia, asthma, multiple sclerosis, type Idiabetes, systemic lupus erythematosus, scleroderma, systemic sclerosis,or Sjögren's syndrome.

In the above-described treatment method, both compounds are preferablyprovided together in a pharmaceutical composition that also includes apharmaceutically acceptable carrier.

The invention also features a pharmaceutical composition that includes(i) a TCA (e.g., amoxapine); and (ii) a corticosteroid (e.g.,prednisolone), along with a pharmaceutically acceptable carrier,diluent, or excipient.

The invention also features a method for identifying compounds usefulfor treating a patient having an inflammatory disorder. The methodincludes the steps of: contacting immune cells in vitro with (i) a TCAor a corticosteroid; and (ii) a candidate compound, and determiningwhether the immune response is modulated relative to (a) immune cellscontacted with the TCA or corticosteroid but not contacted with thecandidate compound, and (b) immune cells contacted with the candidatecompound but not with the TCA or corticosteroid. A candidate compoundthat, when combined with the TCA or corticosteroid, modulates the immuneresponse to a greater degree than controls, is a compound that ispotentially useful for treating a patient having an inflammatorydisorder.

Compounds useful in the invention include those described herein in anyof their pharmaceutically acceptable forms, including isomers such asdiastereomers and enantiomers, salts, solvates, and polymorphs thereof,as well as racemic mixtures of the compounds described herein.

By “tricyclic antidepressant” or “TCA” is meant a compound having onethe formulas (I), (II), or (III):

wherein each X is, independently, H, Cl, F, Br, I, CH₃, CF₃, OH, OCH₃,CH₂CH₃, or OCH₂CH₃; Y is CH₂, O, NH, S(O)₀₋₂, (CH₂)₃, (CH)₂, CH₂O,CH₂NH, CHN, or CH₂S; Z is C or S; A is a branched or unbranched,saturated or monounsaturated hydrocarbon chain having between 3 and 6carbons, inclusive; each B is, independently, H, Cl, F, Br, I, CX₃,CH₂CH₃, OCX₃, or OCX₂CX₃; and D is CH₂, O, NH, S(O)₀₋₂.

In preferred embodiments, each X is, independently, H, Cl, or F; Y is(CH₂)₂, Z is C; A is (CH₂)₃; and each B is, independently, H, Cl, or F.

Exemplary tricyclic antidepressants include, for example, amoxapine,8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine, loxapine succinate,loxapine hydrochloride, 8-hydroxyloxapine, amitriptyline, clomipramine,doxepin, imipramine, trimipramine, desipramine, nortriptyline, andprotriptyline.

By “corticosteroid” is meant any naturally occurring or syntheticsteroid hormone which can be derived from cholesterol and ischaracterized by a hydrogenated cyclopentanoperhydrophenanthrene ringsystem. Naturally occurring corticosteriods are generally produced bythe adrenal cortex. Synthetic corticosteriods may be halogenated.Functional groups required for activity include a double bond at Δ4, aC3 ketone, and a C20 ketone. Corticosteroids may have glucocorticoidand/or mineralocorticoid activity.

Exemplary corticosteroids include, for example, dexamethasone,betamethasone, triamcinolone, triamcinolone acetonide, triamcinolonediacetate, triamcinolone hexacetonide, beclomethasone, dipropionate,beclomethasone dipropionate monohydrate, flumethasone pivalate,diflorasone diacetate, fluocinolone acetonide, fluorometholone,fluorometholone acetate, clobetasol propionate, desoximethasone,fluoxymesterone, fluprednisolone, hydrocortisone, hydrocortisoneacetate, hydrocortisone butyrate, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, hydrocortisone cypionate, hydrocortisone probutate, hydrocortisone valerate, cortisone acetate, paramethasoneacetate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, prednisolone, prednisolone acetate,prednisolone sodium phosphate, prednisolone tebutate, clocortolonepivalate, flucinolone, dexamethasone 21-acetate, betamethasone17-valerate, isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone,dichlorisone, meclorisone, flupredidene, doxibetasol, halopredone,halometasone, clobetasone, diflucortolone, isoflupredone acetate,fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone,fluocinolone, clobetasol, cortisone, paramethasone, clocortolone,prednisolone 21-hemisuccinate free acid, prednisolonemetasulphobenzoate, prednisolone terbutate, and triamcinolone acetonide21-palmitate.

By “low dose corticosteroid” is meant a dose that is less than a dosethat would typically be given to a patient for treatment ofinflammation. Exemplary low doses of corticosteroids are as follows:cortisol: 12 mg/day; cortisone: 15 mg/day; prednisone: 3 mg/day;methylprednisolone: 2.5 mg/day; triamcinolone: 2.5 mg/day;betamethasone: 250 μg/day; dexamethasone: 450 μg/day; hydrocortisone: 9mg/day.

By a “dosage equivalent to an amoxapine dosage” is meant a dosage of aTCA that, in combination with a given dosage of prednisolone, producesthe same anti-inflammatory effect in a patient as a dosage of amoxapinein combination with that dosage of prednisolone.

By a “dosage equivalent to a prednisolone dosage” is meant a dosage of acorticosteroid that, in combination with a given dosage of amoxapine,produces the same anti-inflammatory effect in a patient as a dosage ofprednisolone in combination with that dosage of amoxapine.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

We have discovered that the combination of amoxapine and prednisoloneexhibit substantial TNFα suppressing activity against PBMCs.Concentrations that exhibited effective TNFα suppressing activity werenot unacceptably toxic to normal cells. Thus, this drug combination isuseful for the treatment of an inflammatory disorder.

Amoxapine

Amoxapine is a tricyclic antidepressant (TCA) of the dibenzoxapine type.It is structurally similar to the older TCAs and also sharessimilarities with the phenothiazines.

The exact action of TCAs is not fully understood, but it is believedthat one of their important effects is the enhancement of the actions ofnorepinephrine and serotonin by blocking the reuptake of variousneurotransmitters at the neuronal membrane. Amoxapine also shares somesimilarity with antipsychotic drugs in that it blocks dopamine receptorsand can cause dyskinesia. Amoxapine also blocks the reuptake ofnorepinephrine, similar to the action of desipramine and maprotiline.

Based on the ability of amoxapine to act in concert with prednisolone toinhibit TNFα levels, one skilled in the art will recognize that otherTCAs, as well as structural and functional analogs of amoxapine, canalso be used in combination with prednisolone (or anothercorticosteroid—see below). Amoxapine analogs include, for example,8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine, loxapine succinate,loxapine hydrochloride, 8-hydroxyloxapine, clothiapine, perlapine,fluperlapine, and dibenz (b,f)(1,4)oxazepine,2-chloro-11-(4-methyl-1-piperazinyl)-, monohydrochloride.

Prednisolone

Prednisolone, a synthetic adrenal corticosteroid, has anti-inflammatoryproperties, and is used in a wide variety of inflammatory conditions. Itis desirable to reduce the amount of administered prednisolone becauselong-term use of steroids at can produce significant side effects.

Prednisolone is a member of the corticosteroid family of steroids. Basedon the shared structural features and apparent mechanism of action amongthe corticosteroid family, one skilled in the art will recognize thatother corticosteroids can be used in combination with amoxapine or anamoxapine analog to treat inflammatory disorders. Corticosteroidsinclude, for example, dexamethasone, betamethasone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, triamcinolonehexacetonide, beclomethasone, dipropionate, beclomethasone dipropionatemonohydrate, flumethasone pivalate, diflorasone diacetate, fluocinoloneacetonide, fluorometholone, fluorometholone acetate, clobetasolpropionate, desoximethasone, fluoxymesterone, fluprednisolone,hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate,hydrocortisone sodium phosphate, hydrocortisone sodium succinate,hydrocortisone cypionate, hydrocortisone probutate, hydrocortisonevalerate, cortisone acetate, paramethasone acetate, methylprednisolone,methylprednisolone acetate, methylprednisolone sodium succinate,prednisolone, prednisolone acetate, prednisolone sodium phosphate,prednisolone tebutate, clocortolone pivalate, flucinolone, dexamethasone21-acetate, betamethasone 17-valerate, isoflupredone, 9-fluorocortisone,6-hydroxydexamethasone, dichlorisone, meclorisone, flupredidene,doxibetasol, halopredone, halometasone, clobetasone, diflucortolone,isoflupredone acetate, fluorohydroxyandrostenedione, beclomethasone,flumethasone, diflorasone, fluocinolone, clobetasol, cortisone,paramethasone, clocortolone, prednisolone 21-hemisuccinate free acid,prednisolone metasulphobenzoate, prednisolone terbutate, andtriamcinolone acetonide 21-palmitate.

Therapy

Combination therapy according to the invention may be performed alone orin conjunction with another therapy and may be provided at home, thedoctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment generally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed. The duration of the combination therapy depends on the typeof inflammatory disorder being treated, the age and condition of thepatient, the stage and type of the patient's disease, and how thepatient responds to the treatment. Additionally, a person having agreater risk of developing an inflammatory disorder (e.g., a person whois genetically predisposed or previously had an inflammatory disorder)may receive prophylactic treatment to inhibit or delay an inflammatoryresponse.

The dosage, frequency and mode of administration of each component ofthe combination can be controlled independently. For example, onecompound may be administered orally three times per day, while thesecond compound may be administered intramuscularly once per day.Combination therapy may be given in on-and-off cycles that include restperiods. The compounds may also be formulated together such that oneadministration delivers both compounds.

Formulation of Pharmaceutical Compositions

Suitable modes of administration include oral, rectal, intravenous,intramuscular, subcutaneous, inhalation, topical or transdermal,vaginal, and ophthalmic.

The combination of the invention can also be provided as components of apharmaceutical pack. The two drugs can be formulated together orseparately and in individual dosage amounts.

The compounds of the invention are also useful when formulated as salts.For example, amitriptyline, another tricyclic antidepressant, has beenformulated as a hydrochloride salt, indicating that amoxapine can besimilarly formulated.

Administration of each compound of the combination may be by anysuitable means that results in a concentration of the compound that,combined with the other compound, is anti-inflammatory. Each compound isadmixed with a suitable carrier substance, and is generally present inan amount of 1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable for oral,parenteral (e.g., intravenous, intramuscular, subcutaneous), rectal,transdermal, nasal, vaginal, inhalant, or ocular administration. Thus,the composition may be in form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, deliverydevices, suppositories, enemas, injectables, implants, sprays, oraerosols. The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, (19th ed.) ed. A. R. Gennaro, 1995, MackPublishing Company, Easton, Pa. and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York.

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time period after administration,using controlled release formulations.

Administration of compounds in controlled release formulations is usefulwhere the compound, either alone or in combination, has (i) a narrowtherapeutic index (e.g., the difference between the plasma concentrationleading to harmful side effects or toxic reactions and the plasmaconcentration leading to a therapeutic effect is small; generally, thetherapeutic index, TI, is defined as the ratio of median lethal dose(LD₅₀) to median effective dose (ED₅₀)); (ii) a narrow absorption windowin the gastrointestinal tract; or (iii) a short biological half-life, sothat frequent dosing during a day is required in order to sustain theplasma level at a therapeutic level.

Many strategies can be pursued to obtain controlled release in which therate of release outweighs the rate of metabolism of the therapeuticcompound. For example, controlled release can be obtained by theappropriate selection of formulation parameters and ingredients,including, e.g., appropriate controlled release compositions andcoatings. Examples include single or multiple unit tablet or capsulecompositions, oil solutions, suspensions, emulsions, microcapsules,microspheres, nanoparticles, patches, and liposomes.

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

The two compounds may be mixed together in a tablet or other vehicle, ormay be partitioned. In one example, the first compound is contained onthe inside of the tablet, and the second compound is on the outside,such that a substantial portion of the second compound is released priorto the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

Dosages

The dosage of each compound of the claimed combinations depends onseveral factors, including: the administration method, the disease to betreated, the severity of the disease, whether the disease is to betreated or prevented, and the age, weight, and health of the person tobe treated. Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular patient may affect dosage used.

Continuous daily dosing with the combinations of the invention may notbe required. A therapeutic regimen may require cycles, during which timea drug is not administered, or therapy may be provided on an as neededbasis during periods of acute inflammation.

As described above, the compound in question may be administered orallyin the form of tablets, capsules, elixirs or syrups, or rectally in theform of suppositories. Parenteral administration of a compound issuitably performed, for example, in the form of saline solutions or withthe compound incorporated into liposomes. In cases where the compound initself is not sufficiently soluble to be dissolved, a solubilizer suchas ethanol can be applied.

Below, for illustrative purposes, the dosages for amoxapine andprednisolone are described. One skilled in the art will readily be ableto ascertain suitable dosages for other TCAs and corticosteroids. Forexample, a TCA can be given in a dosage equivalent to an amoxapinedosage provided below, and a corticosteroid can be given in a dosageequivalent to a predinisolone dosage provided below. In one embodiment,the corticosteroid is a low dose corticosteroid.

Oral Administration

For amoxapine adapted for oral administration for systemic use, thetotal daily dosage is normally about 1-600 mg (0.01-8.5 mg/kg),preferably about 25-400 mg (0.35-5.7 mg/kg), and more preferably about100-300 mg (1.4-4.2 mg/kg) total daily dose. Administration can be oneto three times daily for one day to one year, and may even be for thelife of the patient. Chronic, long-term administration will be indicatedin many cases. Daily dosages up to 600 mg may be necessary.

For prednisolone adapted for oral administration for systemic use, thedaily dosage is normally about 0.05-200 mg (0.7-2800 mcg/kg), preferablyabout 0.1-60 mg (1-850 mcg/kg), and more preferably about 0.1-5 mg (4-70mcg/kg). Because of the enhancing effect exhibited by amoxapine onprednisolone anti-inflammatory activity, low dosages of prednisolone(e.g., 0.2, 0.4, 0.6, 0.8, 1, 2, or 3 mg/day), when combined with a TCA,can be effective in treating inflammation. Administration one to fourtimes daily is desirable. Like amoxapine, prednisilone may beadministered for one day to one year, and may even be for the life ofthe patient. Dosages up to 200 mg per day may be necessary.

Rectal Administration

For compositions adapted for rectal use for preventing disease, asomewhat higher amount of a compound is usually preferred. Thus a totaldaily dosage of amoxapine is normally about 1-600 mg (0.01-8.5 mg/kg).Rectal administration of amoxapine is normally one to three times daily.A total daily dosage of prednisolone is normally about 0.1-100 mg(1-1420 mcg/kg). Rectal administration of prednisolone is normally oneto four times daily.

Intravenous Administration

For intravenous administration of amoxapine, a total daily dosage isabout 1-400 mg (0.014-5.7 mg/kg), preferably about 10-200 mg (0.14-2.8mg/kg) and more preferably about 25-100 mg (0.35-1.4 mg/kg). Intravenousadministration of amoxapine is normally one to four times daily, but canbe continuously infused.

For intravenous administration of prednisolone, a total daily dosage isabout 0.05-200 mg (0.0007-2.8 mg/kg), preferably about 0.1-60 mg(0.001-0.85 mg/kg), and more preferably about 0.1-5 mg (4-70 mcg/kg).Low dosages of prednisolone, described above, are most preferred.Intravenous administration of prednisolone is normally one to four timesdaily, but, like amoxapine, can be continuously infused.

Additional Routes of Administration

For intramuscular, subcutaneous, inhalation, topical, vaginal, orophthalmic administration of amoxapine, a total daily dosage is about1-400 mg (0.014-5.7 mg/kg), preferably about 10-200 mg (0.14-2.8 mg/kg),and more preferably about 25-100 mg (0.35-1.4 mg/kg), and a total dailydosage of prednisolone is about 0.1-100 mg (0.0014-1.42 mg/kg). By theseroutes, administration of each of amoxapine and prednisolone is,independently, one to four times daily.

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

EXAMPLE 1 Preparation of Pairwise Compound Mixed Combination SerialDilution Matrix

Stock solutions of amoxapine (16 mg/ml) (Sigma-Aldrich, St. Louis, Mo.;catalog number A129) and prednisolone (1.6 mg/ml) (Sigma-Aldrich,catalog number P6004) were made in dimethylsulfoxide (DMSO). Theamoxapine master plates were made by adding 25 μl of the concentratedstock solution to columns 3, 9, and 15 (rows C through N) of apolypropylene 384-well storage plate that had been pre-filled with 75 μlof anhydrous DMSO. Using a TomTec Quadra Plus liquid handler, the 25 μlof amoxapine stock solution was serially diluted two-fold four timesinto the adjacent columns (columns 4-7, 10-13, 16-19). The sixth column(8, 14, and 20) did not receive any compound and served as a vehiclecontrol. The prednisolone master plates were made by adding 25 μl of theconcentrated prednisolone stock solution to the appropriate wells (rowC, columns 3-8; row C, columns 9-14; row C, columns 15-20; row I,columns 3-8; row I, columns 9-14; row I, columns 15-20) of theappropriate prednisolone master polypropylene 384-well storage plate.These master plates had been pre-filled with 75 μl of anhydrous DMSO.Using the TomTec Quadra Plus liquid handler, the 25 μl was seriallydiluted two-fold four times in the adjacent rows (rows D-G, and J-M).The sixth row (H and N) did not receive any compound to serve as avehicle control. Master plates were sealed and stored at −20 C untilready for use.

The final amoxapine/prednisolone combination plate was generated bytransferring 1 μl from each of the amoxapine and prednisolone masterplates to a dilution plate containing 100 μl of media (RPMI; Gibco BRL,#11875-085), 10% Fetal Bovine Serum (Gibco BRL, #25140-097), 2%Penicillin/Streptomycin (Gibco BRL, #15140-122)) using the TomTec QuadraPlus liquid handler. This dilution plate was then mixed and a 10 μlaliquot transferred to the final assay plate, which had been pre-filledwith 40 μl/well RPMI media containing the appropriate stimulant toactivate TNFα secretion (see below).

EXAMPLE 2 Assay for TNFα Suppressing Activity of Amoxapine andPrednisolone

The compound dilution matrix was assayed using a TNFα ELISA method.Briefly, a 100 μl suspension of diluted human peripheral bloodmononuclear cells (PBMCs) contained within each well of a polystyrene384-well plate (NalgeNunc) was stimulated to secrete TNFα by treatmentwith a final concentration of 10 ng/ml phorbol 12-myristate 13-acetate(Sigma) and 750 ng/μl ionomycin (Sigma). Various concentrations of eachtest compound were added at the time of stimulation. After 16-18 hoursof incubation at 37° C. in a humidified incubator, the plate wascentrifuged and the supernatant transferred to a white opaquepolystyrene 384 well plate (NalgeNunc, Maxisorb) coated with an anti-TNFantibody (PharMingen, #18631D). After a two-hour incubation, the platewas washed (Tecan PowerWasher 384) with phosphate buffered saline (PBS)containing 0.1% Tween. 20 (polyoxyethylene sorbitan monolaurate) andincubated for an additional one hour with another anti-TNF antibody thatwas biotin labeled (PharMingen, 18642D) and horseradish peroxidase (HRP)coupled to strepavidin (PharMingen, #13047E). After the plate was washedwith 0.1% Tween 20/PBS, the HRP substrate (which contains luminol,hydrogen peroxide, and an enhancer such as para-iodophenol) was added toeach well and light intensity measured using a LJL Analyst luminometer.Control wells contained a final concentration of 1 μg/ml cyclosporin A(Sigma).

EXAMPLE 3 Suppression of TNFα Secretion from Phorbol 12-myristate13-acetate and Ionomycin Treated Blood by a Combination of Amoxapine andPrednisolone

Together, amoxapine and prednisolone were able to suppress phorbol12-myristate 13-acetate and ionomycin induced TNFα secretion in blood.As is shown in Tables 1 and 2, amoxapine is able to enhance the doseresponse of prednisolone by nearly two fold. At a concentration of 1.1μM, prednisolone alone is able to inhibit TNFα secretion by 28%.Addition of 0.2 μM amoxapine increases the TNFα inhibition of the 1.11μM prednisolone to 51%. This large increase in activity of 82% iscreated by a relatively small increase of only 18% in total drugspecies.

TABLE 1 Amoxapine [μM] Prednisolone [μM] 12.750 3.188 0.797 0.199 0.0500.000 1.110 85.89 66.47 47.73 50.93 32.79 27.59 0.277 82.82 58.88 48.7934.40 29.17 26.81 0.069 78.58 60.66 41.32 34.99 21.41 22.31 0.017 84.6962.66 34.66 32.48 21.39 19.06 0.004 84.13 53.41 33.86 16.02 5.82 2.690.000 73.02 50.44 24.29 16.66 7.30 0.00

TABLE 2 Amoxapine [μM] Prednisilone [μM] 12.747 6.373 3.187 1.593 0.7970.398 0.199 0.100 0.050 0.000 1.110 88.35 74.63 66.76 65.79 57.72 52.5045.50 43.72 40.04 34.62 0.555 90.09 76.42 69.66 60.08 53.51 46.73 41.7043.67 31.76 30.50 0.277 86.67 75.34 66.45 59.64 54.23 46.97 45.38 35.2034.42 36.89 0.139 91.50 78.45 70.37 60.75 54.73 47.05 41.51 37.79 28.4625.74 0.069 36.59 86.03 77.74 67.81 57.14 49.96 37.24 33.39 31.75 24.560.035 92.76 80.28 70.42 59.40 52.58 47.40 37.94 34.59 21.47 24.06 0.01791.02 75.16 72.06 56.40 45.14 47.84 36.50 24.33 21.92 24.74 0.009 88.5872.16 71.61 56.03 49.80 39.87 28.66 27.23 17.78 14.34 0.004 84.32 66.1457.21 46.53 32.06 27.76 23.73 15.94 12.99 9.62 0.000 80.89 64.40 47.9637.13 21.88 16.38 14.19 3.60 −3.31 −0.97Amoxapine enhancement of prednisolone activity was also observed in afollow-up secondary screen. The TNFα inhibition of prednisolone at aconcentration of 9 nM was increased 2.9 fold to 40% in the presence of400 nM amoxapine. The TNFα inhibition activity of prednisolone andamoxapine alone at these concentrations is only 14 and 16% respectively.Moreover, the level of TNFα inhibition achieved by 9 nM prednisolone incombination with 398 nM amoxapine (40%) is no less than that of 1110 nMprednisolone alone (35%). This increase in TNFα inhibition constitutes apotency shift of as much as 100-fold for the combination, compared toprednisolone alone.

The ability of amoxapine and prednisolone to inhibit TNFα secretion fromLPS stimulated blood is shown in Table 3.

TABLE 3 Amoxapine [μM] Prednisolone [μM] 12.747 6.373 3.187 1.593 0.7970.398 0.199 0.100 0.050 0.000 1.110 78.97 71.52 67.84 63.65 66.04 68.0461.29 64.30 58.19 60.06 0.555 83.61 68.05 62.72 65.82 59.46 56.17 56.3655.47 55.94 47.15 0.277 70.40 64.01 62.08 57.91 55.42 54.64 56.94 51.3950.05 48.75 0.139 72.56 60.77 58.36 55.47 50.42 49.25 49.54 48.74 44.4648.46 0.069 70.27 73.99 61.88 48.82 43.56 47.22 42.13 42.62 44.19 38.790.035 86.37 64.17 43.28 38.16 37.26 37.96 38.06 40.83 32.87 33.11 0.01778.81 48.94 42.94 40.81 37.94 35.96 32.00 35.25 29.35 37.12 0.009 67.0943.76 41.07 34.23 25.54 24.86 31.12 23.57 27.36 30.24 0.004 60.14 37.5934.03 25.52 24.94 27.78 25.57 30.40 18.90 12.06 0.000 49.64 21.26 24.2116.79 13.11 8.10 2.39 5.52 3.00 −1.31

OTHER EMBODIMENTS

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the invention.

1. A composition formulated for topical administration, said compositioncomprising as the sole active ingredients a tricyclic antidepressant anda corticosteroid in amounts that, when administered together to apatient having an immunoinflammatory disorder, inhibit or reduceinflammation or otherwise treat said disorder, wherein said tricyclicantidepressant is selected from the group consisting of naprotiline,amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine,8-hydroxyloxapine, clomipramine, desipramine, imipramine, trimipramine,nortriptyline, and protriptyline.
 2. The composition of claim 1, whereinsaid corticosteroid is in a low dose.
 3. A composition formulated forinhalation administration, said composition comprising a tricyclicantidepressant and a corticosteroid in amounts that, when administeredtogether to a patient having an immunoinflammatory disorder, inhibit orreduce inflammation or otherwise treat said disorder.
 4. The compositionof claim 3, wherein said corticosteroid is in a low dose.
 5. A methodfor treating an immunoinflammatory disorder in patient in need thereof,said method comprising administering to said patient a tricyclicantidepressant and a low dose corticosteroid, wherein the tricyclicantidepressant and the corticosteroid are administered simultaneously,or within 14 days of each other, in amounts that together are sufficientto treat said disorder.
 6. The method of claim 5, wherein said tricyclicantidepressant is administered topically.
 7. The method of claim 5,wherein said immunoinflammatory disorder is selected from the groupconsisting of rheumatoid arthritis, psoriasis, ulcerative colitis,Crohn's disease, stroke-induced brain cell death, ankylosingspondylitis, fibromyalgia, asthma, multiple sclerosis, type I diabetes,systemic lupus erythematosus, scleroderma, systemic sclerosis, andSjögren's syndrome.
 8. The method of claim 5, wherein said tricyclicantidepressant is selected from the group consisting of maprotilinc,amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine,8-hydroxyloxapine, clomipramine, desipramine, doxepin, imipramine,trimipramine, nortriptyline, amitryptiline, and protriptyline.
 9. Themethod of claim 5, wherein said tricyclic antidepressant and saidcorticosteroid are administered simultaneously.
 10. A method fortreating an immunoinflammatory disorder selected from psoriasis,ulcerative colitis, Crohn's disease, stroke-induced brain cell death,ankylosing spondylitis, fibromyalgia, asthma, multiple sclerosis, type Idiabetes, systemic lupus crythemnatosus, sclecroderma, systemicsclerosis, and Sjögren's syndrome in a patient in need thereof; saidmethod comprising administering to said patient a tricyclicantidepressant and a corticosteroid, wherein said tricyclicantidepressant is selected from the group consisting of maprotiline,amoxapine, 8-hydroxyamoxapine, 7-hydroxyamnoxapine, loxapine,8-hydroxyloxapine, amitriplyline, desipramine, clomipramine, doxepin,imipramine, trimipramine, nortriptyline, and protriptyline; wherein thetricyclic antidepressant and the corticosteroid are administeredsimultaneously, or within 14 days of each other, in amounts thattogether are sufficient to treat said disorder.
 11. The method of claim10, whercin said tricyclic antidepressant is administered topically. 12.The method of claim 10, wherein said tricyclic antidepressant and saidcorticosteroid are administered simultaneously.
 13. A method fortreating rheumatoid arthritis in a patient in need thereof, said methodcomprising administering to said patient a tricyclic antidepressant anda corticosteroid, wherein said tricyclic antidepressant is selected fromthe group consisting of maprotiline, amoxapine, 8-hydroxyamoxapine,7-hydroxyamoxapine, loxapine, 8-hydroxyloxapine, clomipramine, doxepin,imipramine, trimipramine, nortriptyline, and protriptyline; wherein thetricyclic antidepressant and the corticosteroid are administeredsimultaneously, or within 14 days of each other, in amounts thattogether are sufficient to treat said rheumatoid arthritis.
 14. Themethod of claim 13, wherein said tricyclic antidepressant isadministered topically.
 15. The method of claim 13, wherein saidtricyclic antidepressant and said corticosteroid are administeredsimultaneously.
 16. A method for treating an immunoinflammatory disorderin patient in need thereof, said method comprising administering to saidpatient a tricyclic antidepresant selected from the group consisting ofmaprotiline, amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine,loxapine, 8-hydroxyloxapine, clomipramine, desipramine, imipramine,trimipramine, nortriptyline, amitriptyline, and protriptyline, and acorticosteroid, wherein said tricyclic antidepressant and thecorticosteroid are administered simultaneously or within 14 days of eachother in amounts that together are sufficient to treat said disorder andsaid tricyclic antidepressant is topically administered to said patient.17. The method of claim 16, wherein said immunoinflammatory disorder isselected from rheumatoid arthritis, psoriasis, ulcerative colitis,Crohn's disease, strokc-induced brain cell death, ankylosingspondylitis, fibromyalgia, asthma, multiple sclerosis, type I diabetes,systemic lupus erythematosus, scleroderma, systemic sclerosis, andSjögren's syndrome.
 18. The method of claim 16, wherein saidcorticosteroid is topically administered to said patient.
 19. The methodof claim 16, wherein said corticosteroid is systemically administered tosaid patient.
 20. The method of claim 16, wherein the corticosteroid isadministered at a low dose.
 21. The method of claim 16, wherein saidtricyclic antidepressant and said corticosteroid are administeredsimultaneously.
 22. A method for treating an immunoinflammatory disorderselected from rheumatoid arthritis, psoriasis, ulcerative colitis,Crohn's disease, stroke-induced brain cell death, ankylosingspondylitis, fibromyalgia, asthma, multiple sclerosis, type I diabetes,systemic lupus crythematosus, scleroderma, systemic sclerosis, andSjögren's syndrome, said method comprising administering to a patient atricyclic antidepressant and a corticosteroid, wherein said tricyclicantidepressant and the corticosteroid are administered simultaneously orwithin 14 days of each other in amounts that together are sufficient totreat said disorder and said tricyclic antidepressant is topicallyadministered to said patient.
 23. The method of claim 22, wherein saidcorticosteroid is topically administered to said patient.
 24. The methodof claim 22, wherein said corticosteroid is systemically administered tosaid patient.
 25. The method of claim 22, wherein the corticosteroid isadministered at a low dose.
 26. The method of claim 22, wherein saidtricyclic antidepressant and sand corticosteroid are administeredsimultaneously.
 27. A method for treating an immunoinflammatory disorderin a patient in need thereof, said method comprising administering tosaid patient a tricyclic antidepressant and a corticosteroid, whereinsaid tricyclic antidepressant and the corticosteroid are administeredsimultaneously or within 14 days of each other in amounts that togetherare sufficient to treat said disorder and said tricyclic antidepressantis administered by inhalation to said patient.
 28. The method of claim27, wherein said corticosteroid is administered by inhalation to saidpatient.
 29. The method of claim 27, wherein the corticosteroid isadministered at a low dose.
 30. The method of claim 27, wherein saidtricyclic antidepressant and said corticosteroid are administeredsimultaneously.
 31. A composition formulated for inhalationadministration, said composition comprising a tricyclic antidepressantand a corticosteroid.
 32. The composition of claim 31, wherein saidtricyclic antidepressant is nortriptyline.
 33. A composition formulatedfor oral administration, said composition comprising a tricyclicantidepressant and a corticosteroid.
 34. The composition of claim 33,wherein said tricyclic antidepressant is nortriptyline.
 35. A method fortreating asthma in a patient in need thereof, said method comprisingadministering to said patient a composition comprising a tricyclicantidepressant and a corticosteroid, wherein said composition isformulated for inhalation administration.
 36. The method of claim 35,wherein said tricyclic antidepressant is nortriptyline.
 37. A method fortreating asthma in a patient in need thereof, said method comprisingadministering to said patient a composition comprising a tricyclicantidepressant and a corticosteroid, wherein said composition isformulated for oral administration.
 38. The method of claim 37, whereinsaid tricyclic antidepressant is nortriptyline.
 39. A compositionformulated for oral, rectal, intravenous, intramuscular, subcutaneous,inhalation, vaginal, or ophthalmic administration, said compositioncomprising a tricyclic antidepressant and a corticosieroid, wherein saidtricyclic antidepressant is selected from the group consisting ofmaprotiline, amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine,loxapine, 8-hydroxyloxapine, clomipramine, desipramine, imipramine,trimipramine, nortriptyline, amitriptyline, and protriptyline.
 40. Thecomposition of claim 39, wherein said tricyclic antidepressant ispresent at a dosage between 1-600 mg.
 41. The composition of claim 40,wherein said tricyclic antidepressant is present at a dosage between1-400 mg.
 42. The composition of claim 41, wherein said tricyclicantidepressant is present at a dosage between 1-300 mg.
 43. Thecomposition of claim 39, wherein said corticosteroid is present at adosage between 0.05-200 mg.
 44. The composition of claim 43, whereinsaid corticosteroid is present at a dosage between 0.1-60 mg.
 45. Thecomposition of claim 44, wherein said corticosteroid is present at adosage between 0.1-5 mg.
 46. The composition of claim 39, wherein saidtricyclic antidepressant is present at a dosage equivalent to a dosageof amoxapine between 1-600 mg.
 47. The composition of claim 46, whereinsaid tricyclic antidepressant is present at a dosage equivalent to adosage of amoxapine between 1-400 mg.
 48. The composition of claim 47,wherein said tricyclic antidepressant is present at a dosage equivalentto a dosage of amoxapine between 1-300 mg.
 49. The composition of claim39, wherein said corticosteroid is present at a dosage equivalent to adosage of prednisolone between 0.05-200 mg.
 50. The composition of claim49, wherein said corticosteroid is present at a dosage equivalent to adosage of prednisolone between 0.1-60 mg.
 51. The composition of claim50, wherein said corticosteroid is present at a dosage equivalent to adosage of prednisolone between 0.1-5 mg.
 52. A composition formulatedfor oral, rectal, intravenous, intramuscular, subcutaneous, inhalation,vaginal, or ophthalmic administration, said composition comprising atricyclic antidepressant and a corticosteroid, wherein said tricyclicantidepressant is selected from the group consisting of maprotiline,amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine,8-hydroxyloxapine, clomipramine, desipramine, imipramine, trimipramine,nortriptyline, amitriptyline, and protriptyline; wherein said tricyclicantidepressant is present at a dosage between 1-300 mg; and wherein saidcorticosteroid is present at a dosage between 0.1-60 mg.
 53. Thecomposition of claim 52, wherein said corticosteroid is present at adosage between 0.1-5 mg.
 54. A composition formulated for oral, rectal,intravenous, intramuscular, subcutaneous, inhalation, vaginal, orophthalmic administration, said composition comprising a tricyclicantidepressant and a corticosteroid, wherein said tricyclicantidepressant is selected from the group consisting of maprotiline,amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine,8-hydroxyloxapine, clomipramine, desipramine, imipramine, trimipramine,nortriptyline, amitriptyline, and protriptyline; wherein said tricyclicantidepressant is present at a dosage equivalent to a dosage ofamoxapine between 1-300 mg; and wherein said corticosteroid is presentat a dosage equivalent to a dosage of prednisolone between 0.1-60 mg.55. The composition of claim 54, wherein said corticosteroid is presentat a dosage equivalent to a dosage of prednisolone between 0.1-5 mg. 56.A composition formulated for topical administration, said compositioncomprising as the sole active ingredients a tricyclic antidepressant anda corticosteroid, wherein said tricyclic antidepressant is selected fromthe group consisting of maprotiline, amoxapine, 8- hydroxyamoxapine,7-hydroxyamoxapine, loxapine, 8-hydroxyloxapine, clomipramine,desipramine, imipramine, trimipramine, nortriptyline, and protriptyline.57. The composition of claim 34, wherein said corticosteroid isprednisolone.
 58. The composition of claim 57, wherein said tricyclicantidepressant is present at a dosage between 1-300 mg and saidcorticosteroid is present at a dosage between 0.1-60 mg.
 59. Thecomposition of claim 58, wherein said corticosteroid is present at adosage between 0.1-5 mg.